The long-term goal of the proposal is to develop an effective carbohydrate-based vaccine against HIV-1. The HIV-1 envelope glycoprotein gp120 is heavily glycosylated and carbohydrates constitute approximately 50% of its molecular weight. Despite their abundance on HIV-1 surface, carbohydrate antigens have not yet been adequately exploited for HIV-1 vaccine design. Recent studies revealed that HIV-1 gp120 presents a high density of high-mannose type N-glycans and most of the N-glycosylation sites are conserved among HIV-1 isolates, even though some are located at the hypervariable regions of gp120. Molecular modeling studies indicate that the high-mannose oligosaccharides are clustered to form an oligosaccharide microdomain on gp120. Epitope mapping demonstrated that a high-mannose oligosaccharide cluster most likely constitutes the actual epitopes of the broadly neutralizing antibody 2G12. Taken together, these data suggest that the high-mannose oligosaccharide microdomain provides a unique, conserved antigenic structure for anti-HIV vaccine design. We hypothesize that immunogens incorporating the unique high-mannose microdomain structure will elicit antibodies capable of broadly neutralizing HIV-1 primary isolates. To test this hypothesis, we will perform experiments described in two specific aims. In specific aim 1, we will synthesize novel high-mannose type oligosaccharide clusters that will mimic the oligosaccharide microdomain antigenic structure on HIV-1. Man9GlcNAc2 will be chosen as the high-mannose oligosaccharide moiety and monosaccharides will be used as the scaffold (template) to present these oligosaccharides to form a clustering structure. The high-mannose oligosaccharide clusters will then be conjugated to KLH, an immune-stimulating carrier protein, to fashion a functional immunogen. In specific aim 2, we will test the hypothesis that immunization with the synthetic conjugate-vaccine will raise neutralizing antibody responses against HIV-1 primary isolates. The synthetic conjugate-vaccine will be used to immunize mice. The immune responses will be analyzed using ELISAs and virus neutralization assays. [unreadable] [unreadable] [unreadable]